In this renewal application I propose experiments to study how a neuroinvasive alpha herpesvirus (pseudorabies virus, PRV) infection drives axonal targeting of virions and viral proteins to promote anterograde spread of infection in the nervous system. These experiments take advantage of our technology advances in imaging, neuronal culturing systems, mass spectrometry, and deep sequencing, as well as new findings from the last funding period. In particular, we will focus on imaging US9, a critical protein for sorting virions into axons (aim 1). Using mass spectrometry and new enrichment techniques, we will determine the proteome of enriched vesicles containing GFP-US9 and gE-GFP and initiate studies to identify US9-specific protein complexes (aim 2). Finally, we will extend our work to test new hypotheses concerning the axonal damage response, the role of local axonal translation on viral axonal transport, and, using deep sequencing technology (Ilumina platform), how PRV infection affects not only axonal targeting of viral proteins, but also of host and viral mRNAs (aim 3).